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Potential immunocompetence of proteolytic fragments produced by proteasomes before evolution of the vertebrate immune system.

Niedermann G, Grimm R, Geier E, Maurer M, Realini C, Gartmann C, Soll J, Omura S, Rechsteiner MC, Baumeister W, Eichmann K - J. Exp. Med. (1997)

Bottom Line: Unexpectedly, we found that several high copy ligands of MHC class I molecules, in particular, self-ligands, are major products in digests of source polypeptides by invertebrate proteasomes.However, these changes are quantitative and do not confer qualitatively novel characteristics to proteasomal proteolysis.The data suggest that proteasomes may have influenced the evolution of MHC class I molecules.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck-Institut für Immunbiologie, 79108 Freiburg, Germany.

ABSTRACT
To generate peptides for presentation by major histocompatibility complex (MHC) class I molecules to T lymphocytes, the immune system of vertebrates has recruited the proteasomes, phylogenetically ancient multicatalytic high molecular weight endoproteases. We have previously shown that many of the proteolytic fragments generated by vertebrate proteasomes have structural features in common with peptides eluted from MHC class I molecules, suggesting that many MHC class I ligands are direct products of proteasomal proteolysis. Here, we report that the processing of polypeptides by proteasomes is conserved in evolution, not only among vertebrate species, but including invertebrate eukaryotes such as insects and yeast. Unexpectedly, we found that several high copy ligands of MHC class I molecules, in particular, self-ligands, are major products in digests of source polypeptides by invertebrate proteasomes. Moreover, many major dual cleavage peptides produced by invertebrate proteasomes have the length and the NH2 and COOH termini preferred by MHC class I. Thus, the ability of proteasomes to generate potentially immunocompetent peptides evolved well before the vertebrate immune system. We demonstrate with polypeptide substrates that interferon gamma induction in vivo or addition of recombinant proteasome activator 28alpha in vitro alters proteasomal proteolysis in such a way that the generation of peptides with the structural features of MHC class I ligands is optimized. However, these changes are quantitative and do not confer qualitatively novel characteristics to proteasomal proteolysis. The data suggest that proteasomes may have influenced the evolution of MHC class I molecules.

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Proteasomes from eukaryotic invertebrates have the capacity  to efficiently generate known MHC class I ligands. (A) Digestion of the  44-mer Ova239-281 with 20S proteasomes isolated from D. melanogaster  Schneider cells. The proteasome cleavage sites indicated by the arrows above  the sequence were determined by Edman degradation pool sequencing  (for raw data see Fig. 5 B). Reverse phase HPLC chromatogram of the  peptide mixture is shown below. The mass spectrometry inset refers to  the peak that contains the immunodominant CTL epitope SIINFEKL. (B  and C) Digestion of the 24-mer BTG197-120 with 20S proteasomes isolated from S. cerevisiae. (B) Proteasome cleavage site determination by Edman degradation pool sequencing. For interpretation of pool-sequencing  data, see legend to Fig. 5. (C) Reverse phase HPLC chromatogram. The  numbered peaks contain the peptides LLPSEL (1), TLWVDPYE (2), and  the HLA A2.1 ligand TLWVDPYEV (3). (D) Digestion of the 21-mer  JAK1348-368 with 20S proteasomes isolated from D. melanogaster Schneider  cells. Proteasome cleavage sites indicated by the arrows above the sequence were determined from the peptide products identified in the reverse phase HPLC chromatogram shown below. The numbered peaks  contain the peptides REEWNNF (1), REEWNNFSY (2), SYFPEI (3),  and the Kd ligand SYFPEITHI (4). All peptide mixtures shown were analyzed after substrate consumption. The peptides contained in the peaks  marked with numbers and/or mass spectrometry inserts were identified  by Maldi-Tof-MS (insets) and Edman degradation (not shown).
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Figure 3: Proteasomes from eukaryotic invertebrates have the capacity to efficiently generate known MHC class I ligands. (A) Digestion of the 44-mer Ova239-281 with 20S proteasomes isolated from D. melanogaster Schneider cells. The proteasome cleavage sites indicated by the arrows above the sequence were determined by Edman degradation pool sequencing (for raw data see Fig. 5 B). Reverse phase HPLC chromatogram of the peptide mixture is shown below. The mass spectrometry inset refers to the peak that contains the immunodominant CTL epitope SIINFEKL. (B and C) Digestion of the 24-mer BTG197-120 with 20S proteasomes isolated from S. cerevisiae. (B) Proteasome cleavage site determination by Edman degradation pool sequencing. For interpretation of pool-sequencing data, see legend to Fig. 5. (C) Reverse phase HPLC chromatogram. The numbered peaks contain the peptides LLPSEL (1), TLWVDPYE (2), and the HLA A2.1 ligand TLWVDPYEV (3). (D) Digestion of the 21-mer JAK1348-368 with 20S proteasomes isolated from D. melanogaster Schneider cells. Proteasome cleavage sites indicated by the arrows above the sequence were determined from the peptide products identified in the reverse phase HPLC chromatogram shown below. The numbered peaks contain the peptides REEWNNF (1), REEWNNFSY (2), SYFPEI (3), and the Kd ligand SYFPEITHI (4). All peptide mixtures shown were analyzed after substrate consumption. The peptides contained in the peaks marked with numbers and/or mass spectrometry inserts were identified by Maldi-Tof-MS (insets) and Edman degradation (not shown).

Mentions: We have previously shown that the immunodominant ovalbumin epitope Ova257-264 (SIINFEKL; reference 30) is the major stable product generated by mouse 20S proteasomes from the 22-mer OvaY249-269 as well as from the 44-mer Ova239-281 (12). Here we show that this octamer is also a dominant product of digestion of Ova239-281 by 20S proteasomes isolated from D. melanogaster Schneider cells (Fig. 3 A and Fig. 4 A). As a second example, we studied the generation of the nonamer TLWVDPYEV, an endogenous peptide derived from the product of the antiproliferative B cell translocation gene 1 (BTG1) and eluted as a major self-epitope from the human class I molecule HLA-A2.1 (31). Fig. 3, B and C show that this nonamer peptide is the major dual cleavage product generated by yeast (S. cerevisiae) proteasomes of the synthetic 24 mer encompassing this peptide in the sequence of BTG1. Moreover, we studied a 21-mer sequence derived from the tyrosine kinase JAK1 containing the nonamer SYFPEITHI, the most abundant self-peptide presented by mouse H-2Kd molecules of P815 cells (32, 33), and previously shown to be generated by digestion with mouse 20S proteasomes (34). We detected the epitope as the predominant dual cleavage product of the 21 mer with Drosophila proteasomes (Fig. 3 D). Thus, proteasomes from invertebrate eukaryotes have a high potency to generate proteolytic fragments that have been proven to serve as ligands of MHC molecules in the vertebrate immune system.


Potential immunocompetence of proteolytic fragments produced by proteasomes before evolution of the vertebrate immune system.

Niedermann G, Grimm R, Geier E, Maurer M, Realini C, Gartmann C, Soll J, Omura S, Rechsteiner MC, Baumeister W, Eichmann K - J. Exp. Med. (1997)

Proteasomes from eukaryotic invertebrates have the capacity  to efficiently generate known MHC class I ligands. (A) Digestion of the  44-mer Ova239-281 with 20S proteasomes isolated from D. melanogaster  Schneider cells. The proteasome cleavage sites indicated by the arrows above  the sequence were determined by Edman degradation pool sequencing  (for raw data see Fig. 5 B). Reverse phase HPLC chromatogram of the  peptide mixture is shown below. The mass spectrometry inset refers to  the peak that contains the immunodominant CTL epitope SIINFEKL. (B  and C) Digestion of the 24-mer BTG197-120 with 20S proteasomes isolated from S. cerevisiae. (B) Proteasome cleavage site determination by Edman degradation pool sequencing. For interpretation of pool-sequencing  data, see legend to Fig. 5. (C) Reverse phase HPLC chromatogram. The  numbered peaks contain the peptides LLPSEL (1), TLWVDPYE (2), and  the HLA A2.1 ligand TLWVDPYEV (3). (D) Digestion of the 21-mer  JAK1348-368 with 20S proteasomes isolated from D. melanogaster Schneider  cells. Proteasome cleavage sites indicated by the arrows above the sequence were determined from the peptide products identified in the reverse phase HPLC chromatogram shown below. The numbered peaks  contain the peptides REEWNNF (1), REEWNNFSY (2), SYFPEI (3),  and the Kd ligand SYFPEITHI (4). All peptide mixtures shown were analyzed after substrate consumption. The peptides contained in the peaks  marked with numbers and/or mass spectrometry inserts were identified  by Maldi-Tof-MS (insets) and Edman degradation (not shown).
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Figure 3: Proteasomes from eukaryotic invertebrates have the capacity to efficiently generate known MHC class I ligands. (A) Digestion of the 44-mer Ova239-281 with 20S proteasomes isolated from D. melanogaster Schneider cells. The proteasome cleavage sites indicated by the arrows above the sequence were determined by Edman degradation pool sequencing (for raw data see Fig. 5 B). Reverse phase HPLC chromatogram of the peptide mixture is shown below. The mass spectrometry inset refers to the peak that contains the immunodominant CTL epitope SIINFEKL. (B and C) Digestion of the 24-mer BTG197-120 with 20S proteasomes isolated from S. cerevisiae. (B) Proteasome cleavage site determination by Edman degradation pool sequencing. For interpretation of pool-sequencing data, see legend to Fig. 5. (C) Reverse phase HPLC chromatogram. The numbered peaks contain the peptides LLPSEL (1), TLWVDPYE (2), and the HLA A2.1 ligand TLWVDPYEV (3). (D) Digestion of the 21-mer JAK1348-368 with 20S proteasomes isolated from D. melanogaster Schneider cells. Proteasome cleavage sites indicated by the arrows above the sequence were determined from the peptide products identified in the reverse phase HPLC chromatogram shown below. The numbered peaks contain the peptides REEWNNF (1), REEWNNFSY (2), SYFPEI (3), and the Kd ligand SYFPEITHI (4). All peptide mixtures shown were analyzed after substrate consumption. The peptides contained in the peaks marked with numbers and/or mass spectrometry inserts were identified by Maldi-Tof-MS (insets) and Edman degradation (not shown).
Mentions: We have previously shown that the immunodominant ovalbumin epitope Ova257-264 (SIINFEKL; reference 30) is the major stable product generated by mouse 20S proteasomes from the 22-mer OvaY249-269 as well as from the 44-mer Ova239-281 (12). Here we show that this octamer is also a dominant product of digestion of Ova239-281 by 20S proteasomes isolated from D. melanogaster Schneider cells (Fig. 3 A and Fig. 4 A). As a second example, we studied the generation of the nonamer TLWVDPYEV, an endogenous peptide derived from the product of the antiproliferative B cell translocation gene 1 (BTG1) and eluted as a major self-epitope from the human class I molecule HLA-A2.1 (31). Fig. 3, B and C show that this nonamer peptide is the major dual cleavage product generated by yeast (S. cerevisiae) proteasomes of the synthetic 24 mer encompassing this peptide in the sequence of BTG1. Moreover, we studied a 21-mer sequence derived from the tyrosine kinase JAK1 containing the nonamer SYFPEITHI, the most abundant self-peptide presented by mouse H-2Kd molecules of P815 cells (32, 33), and previously shown to be generated by digestion with mouse 20S proteasomes (34). We detected the epitope as the predominant dual cleavage product of the 21 mer with Drosophila proteasomes (Fig. 3 D). Thus, proteasomes from invertebrate eukaryotes have a high potency to generate proteolytic fragments that have been proven to serve as ligands of MHC molecules in the vertebrate immune system.

Bottom Line: Unexpectedly, we found that several high copy ligands of MHC class I molecules, in particular, self-ligands, are major products in digests of source polypeptides by invertebrate proteasomes.However, these changes are quantitative and do not confer qualitatively novel characteristics to proteasomal proteolysis.The data suggest that proteasomes may have influenced the evolution of MHC class I molecules.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck-Institut für Immunbiologie, 79108 Freiburg, Germany.

ABSTRACT
To generate peptides for presentation by major histocompatibility complex (MHC) class I molecules to T lymphocytes, the immune system of vertebrates has recruited the proteasomes, phylogenetically ancient multicatalytic high molecular weight endoproteases. We have previously shown that many of the proteolytic fragments generated by vertebrate proteasomes have structural features in common with peptides eluted from MHC class I molecules, suggesting that many MHC class I ligands are direct products of proteasomal proteolysis. Here, we report that the processing of polypeptides by proteasomes is conserved in evolution, not only among vertebrate species, but including invertebrate eukaryotes such as insects and yeast. Unexpectedly, we found that several high copy ligands of MHC class I molecules, in particular, self-ligands, are major products in digests of source polypeptides by invertebrate proteasomes. Moreover, many major dual cleavage peptides produced by invertebrate proteasomes have the length and the NH2 and COOH termini preferred by MHC class I. Thus, the ability of proteasomes to generate potentially immunocompetent peptides evolved well before the vertebrate immune system. We demonstrate with polypeptide substrates that interferon gamma induction in vivo or addition of recombinant proteasome activator 28alpha in vitro alters proteasomal proteolysis in such a way that the generation of peptides with the structural features of MHC class I ligands is optimized. However, these changes are quantitative and do not confer qualitatively novel characteristics to proteasomal proteolysis. The data suggest that proteasomes may have influenced the evolution of MHC class I molecules.

Show MeSH